Method and device for carrying out chromatographic analyses in an automatic and optimised manner

ABSTRACT

The invention relates to a method for automatically carrying out chromatographic analyses. According to said method, at least one of the following pieces of information is obtained: a) the retention times of at least one constituent; b) the temperature; c) the structural formulae of at least one of the constituents; d) the type of elution agent; and e) the parameters of the separation column. For extensive optimisation, the invention is characterised in that the data obtained is optionally completed by available data, at least one first test run is carried out under isocratic conditions, and retention times are detected or calculated on the basis of a model with optimisation parameters. The retention times obtained are optimised by varying the parameters, another chromatography run is carried out with the parameters of the optimised retention times, and a comparison is made with the measured retention times. The above-mentioned steps are optionally repeated for further optimisation.

[0001] The present invention relates to a method and a device for theautomatic performance and optimization of chromatographic investigationswith the help of an electronic data processing apparatus, the methodhaving the following steps:

[0002] i) Recording of data which include at least one of the followingitems of information

[0003] a) Retention times of at least one component of the substancemixture, preferably for at least two different concentrations of anorganic modifier,

[0004] b) The temperature at which the retention times were ascertained,

[0005] c) Structural formulae of components of the substance mixture,

[0006] d) Type of eluent and concentration of an organic modifiertherein,

[0007] e) Parameters and/or sort and type of separation column.

[0008] The corresponding device has a chromatographic column orseparation column, an inlet chamber for the substance mixture, an eluentfeed, a measuring unit at the outlet of the chromatography column andalso a data-recording unit for the automatic and/or manual inputting ofdata.

[0009] Corresponding methods and devices are known in the state of theart and are offered for sale and marketed for example by Merck KGaAunder the name “LaChrom”.

[0010] In principle, the known systems work reliably and quickly if theanalytes, i.e. the individual components of the substance mixture, areadequately known. If it is for example a matter of confirming and/orexcluding a specific composition of a substance mixture, such as e.g. ofa drug, i.e. that e.g. a certain undesired by-product which can easilyform in the preparation process is contained in the mixture, specificchromatography conditions can be set for this as standard on the basisof empirical values, and at least partly also allowed to proceedautomatically. As the sought components, just like any undesiredcomponents, are known, the individual bands can be clearly allocated tothe components using the chromatogram which was produced based oncorresponding empirical values, quantitative estimates also beingpossible due to the surface area given below the bands.

[0011] The parameters to be modified are for example the temperature andthe composition of the eluent. In the case of so-called “reversed-phasechromatography”, which is the most important in practice, the eluent canbe for example a mixture of water and a non-polar solvent, such as e.g.methanol or acetonitrile. The non-polar solvent is also called “organicmodifier” in technical language. The so-called “stationary phase”, i.e.the material contained in the chromatography column, is in general ahighly porous solid or a packing of solid granules or particles, thesurface of this solid phase likewise being non-polar and for thispurpose optionally being coated with a non-polar material. The analytesaccumulate on the surface of the solid phase and the through-washingand/or through-pressing of the eluent through the chromatography columnhas the result that within a certain period, a component is detachedfrom the surface of the solid phase and discharged with the eluent atthe outlet of the chromatography column. The time which elapses from thebeginning of the chromatography run to the appearance of a component atthe outlet of the chromatography column is called the “retention time”of this component. An essential parameter influencing the retentiontimes in the chromatography column is the mixing ratio between water andorganic modifier or, in other words, the concentration of the organicmodifier in the eluent. Finally, as a further parameter, the material,i.e. the surface composition, and also the porosity and/or the grainsize of the column material, play an important part.

[0012] The substance mixture introduced into an inlet chamber is firstlyextensively adsorbed by the column material. The eluent then detaches,depending on the interaction with the individual components and theirinteraction with the column, the individual components from the columnmaterial and transports them gradually to the outlet of thechromatography column. Due to the different solubilities and interactionenergies between the individual analytes and the eluent on the one handand between the analytes and the column material on the other, thecomponents are detached at different speeds in the eluent and inasmuchare also transported through and out of the column at different speeds.This leads to a separation of the individual components from one anotherwhich are transported consecutively out of the outlet of the column andthrough the measuring cell. If the solubilities at a given concentrationare very different, this bears on the duration of the chromatographyrun, because the portion of better-soluble components possibly appearsrelatively quickly at the outlet of the column whereas verypoorly-soluble components take an extremely long time until they havebeen detached by the eluent and transported to the outlet of the column.In this case, the chromatogram, which is e.g. nothing other than the UVabsorption in the cell as a function of time, can be very prolonged andthus require a very great deal of time, in addition to which the bandsor peaks which appear very late can also be markedly flattened and thusscarcely recognizable.

[0013] This is encountered in general by increasing the concentration ofthe so-called organic modifier. Increasing the concentration of theorganic modifier increases the probability of the components of thesubstance mixture being separated from the chromatography column andtransported by the eluent. However, this can also result in differentcomponents appearing almost simultaneously at the outlet of thechromatography column, so that the corresponding bands can no longer beclearly separated from one another and in particular the surface belowthe bands can also no longer be sufficiently precisely recorded.

[0014] While the correct parameters can be set without major problems inthe case of routine chromatography runs with which for example merely aconsistent quality of a specific product is to be ensured, the settingof the parameters in the case of substance mixtures whose composition isnot, or only partly, known represents a considerable problem. With knownsubstances or routine investigations, the optimum parameters areselected on the basis of empirical values, i.e. essentially the columnmaterial, the temperature to be set and the concentration of the organicmodifier.

[0015] Parameters are regarded as “optimum” which result in eachcomponent of the substance mixture being individually recognizable asseparate bands clearly separated from the bands produced by othercomponents on the chromatogram, and furthermore the time requiredoverall for a chromatography run is as short as possible.

[0016] The “ideal” chromatogram consequently consists of a series ofbands in close succession which are also fully separated from oneanother, where the first band should appear as early as possible afterthe start of the chromatography run. The “bands” or “peaks” can beproduced for example by UV absorption, the eluent leaving thechromatography column being guided, together with the material dissolvedtherein, through a measuring cell which is irradiated from one side withUV light, while on the opposite side of the cell there is arranged adetector for UV light with the help of which the UV absorption of thecomponents, which are contained in the eluent flowing though the cell,of the substance mixture can be recorded. Naturally other detectionmethods for the individual, dissolved components are also conceivable,including mass-spectrometry investigations.

[0017] In order to avoid both too long a duration for the chromatogramand a defective band separation, in many cases the concentration of theorganic modifier is varied during the chromatography run (so-calledgradient elution chromatography). At the start there is generally a lowconcentration of organic modifier, which is e.g. increased continuouslyuntil the end of the chromatography run. It is however often expedientnot to leave the concentration gradient constant, but vary it moremarkedly and e.g. to change the concentration in stages or also to onlychange it continuously for a limited time and otherwise to proceed atconstant concentrations.

[0018] Because in addition the temperature is also available as avariation parameter, the control of chromatography runs becomesincreasingly more complicated the more components are contained in asubstance mixture and the less that is known about the substancemixture. With largely unknown substance mixtures, frequently expensiveand lengthy test runs are required, the user of such a chromatographyapparatus deciding, on the basis of intermediate results and on thebasis of possibly many years' experience which parameters are to bechanged and when. This is not only a very time-consuming process whichsimultaneously also requires a very great amount of manpower, but ismoreover also a procedure which is very prone to errors, as thechanging, based on intuition and experience, of parameters, which isoften carried out in order to limit the long duration of achromatography run and to accelerate the process, very often results inbands coinciding unnoticed so that certain components are not identifiedor found at all.

[0019] In the state of the art the method has thus far been automatedinasmuch as the user of the chromatography apparatus selects and inputsa set of parameters and the chromatography apparatus then automaticallycarries out the chromatography runs with the selected parameters. Forthis, very many test runs are usually necessary and not all the selectedparameters lead to meaningful results. The best results must be readmanually from the obtained chromatograms.

[0020] Compared with this state of the art, the object of the presentinvention is to create a method and a device for chromatographyinvestigations which require less manpower and yet can ensure a fullrecording of all components, or as many components as possible, of thesubstance mixture in a relatively short time.

[0021] This object is achieved by a method according to claim 1 and adevice according to claim 16.

[0022] The method according to the invention proposes in general thefollowing steps:

[0023] i) Recording of data which include at least one of the followingitems of information

[0024] a) Retention times of at least one component of the substancemixture, preferably for at least two different concentrations of anorganic modifier,

[0025] b) The temperature at which the retention times were ascertained,

[0026] c) Structural formulae of one or more of the components of theanalyte,

[0027] d) Type of eluent and also the concentration of an organicmodifier therein,

[0028] e) Parameters or type of separation column,

[0029] the recorded data being fully or partly able to also be retrievedand/or supplemented from a data storage apparatus and, where data areunavailable for the items of information according to a), b) or c), atleast a first test run taking place under isocratic conditions in orderto record retention times according to a), and preferably a furtherisocratic test run being carried out with a different concentration ofthe organic modifier,

[0030] ii) Calculation of retention times as a function of settableparameters based on a model which reproduces a functional relationshipof at least part of the data recorded under i), the recorded data alsobeing able to be reproduced by setting model parameters,

[0031] iii) Optimization of the retention times in the model by varyingthe concentration of the organic modifier and/or of the temperature,

[0032] iv) Carrying out at least one further chromatography run with theparameters of the retention times optimized according to step iii), andcomparing the actual retention times with the theoretically calculated,optimized retention times,

[0033] v) Optionally single or repeated repetition of steps i) to iv) ifthe deviations of the calculated and the measured retention times exceeda presettable limit value or if the differences between the retentiontimes of two successive chromatography runs fall short of a presettablelimit value,

[0034] all above-named steps, with the exception of an initial datainput, proceeding automatically and independently under the control of adata processing apparatus with an artificial intelligence module.

[0035] It is known to a person skilled in the art that retention timescan also be influenced by other parameters such as e.g. pH value orionic strength. According to the invention, these parameters arecollectively called optimizing parameters. If the substance mixtureconsists only of a few components, the retention times of which atspecific concentrations of the organic modifier are known, it is ingeneral relatively simple to set the concentration, the concentrationpattern and/or the temperature such that each of the components has aclearly distinct retention time with the result that the correspondingpeaks of the chromatogram are cleanly separated from one another and yetsimultaneously the longest retention time of the components is kept asshort as possible.

[0036] If however the number of components is relatively large and theretention times have different, in some cases clearly distinct,concentration and/or temperature relationships, the whole system veryquickly becomes difficult to oversee and can be optimized only with luckeven with a great deal of experience by decisions of the user. Accordingto the present invention, it is proposed for this case to carry out anoptimization with the help of mathematical procedures by choosing theretention times as functions of the settable variables in a model, i.e.based on a model which contains at least part of the available data asparameters or measured values. By varying the concentration of theorganic modifier and/or of also the temperature in the model, theretention times are subsequently optimized, it being understood that theretention times actually measured at given concentrations must bereproduced correctly in the model. This is done in general by adaptingmodel parameters which are assumed to be constant for furtheroptimization. An artificial intelligence module ultimately decides whichparameters are regarded as optimum and sends corresponding instructionsto a control unit.

[0037] The more difficult application of the method according to theinvention occurs if only a few components of the substance mixture areknown, but the majority are not known. For these components, eitherspecific retention times at certain concentrations of the organicmodifier can be known or alternatively or in addition the structuralformulae of components of the substance mixture. Based on these data, atheoretical model is again taken as a basis and with this theoreticalmodel the retention times of all the known components at variousconcentrations are calculated and the concentrations of the organicmodifier optimized as a function of time. The temperature can also beused as an additional optimizing parameter, in particular e.g. if twocomponents are difficult to separate from one another at a giventemperature, yet at the same time their retention times display aclearly distinct temperature pattern.

[0038] If no data whatsoever are available regarding structural formulaeor retention times, a test run is simply carried out under isocraticconditions, i.e. at a constant concentration of the organic modifier.

[0039] Preferably a second test run is also carried out at a secondconcentration of the organic modifier. Usually at least some bandsappear at both concentrations in the respective chromatogram. Startingfrom the time of the introduction of a substance mixture into a suitableinlet chamber and the inputting of the optionally previously availabledata, the process runs fully automatically and the corresponding deviceis designed for such an automatic process, i.e. in addition to the basicconstituents of a chromatography column with inlet chamber for thesubstance mixture, an eluent feed and the measuring unit at the outletof the chromatography column, it also has a data-recording apparatus forthe automatic and/or manual inputting of data, an associatedoptimization unit which calculates, based on the most recently recordeddata of a system, optimized parameters for a (further) chromatographyrun and a control apparatus and also parameter sensors which areprovided to automatically set the parameters ascertained in theoptimization unit and to control the parameters according to theautomatically ascertained process.

[0040] If two test runs have been carried out, it can be attempted toassign measured bands of the two chromatograms to each other or to aspecific component. A clear assignment is achieved by using standardsfor each individual component of the substance mixture and carrying outthe experiments only with the respective individual standards. Standardsof the individual components with a purity >80% are preferably used forthis. The method can be further improved by using intelligentpeak-tracking strategies. In the preferred version, a suitable modelexpediently assumes that the retention times decrease with increasingconcentration of the organic modifier. The surface present below a bandof the chromatogram offers further assistance. This naturally assumesthat identical quantities of the substance mixture are introduced intothe chromatography column each time or that at least the introducedquantities are precisely known and the substance mixture has a constantcomposition. An assignment of the peaks can also be carried out viaspectral properties of the components. As a result of thesepeak-tracking strategies, the number of standards required can bereduced to two and the optimizing experiments carried out both firstlywith the standards and also thereafter with the substance mixture.

[0041] The optimization unit then again calculates, on the basis of therecorded concentration pattern of the retention times for the differentcomponents of the system, optimized parameters which are set in afurther chromatography run. The retention times actually measured arecompared with the previously calculated times and the model parameterstaken as a basis are adapted such that the model now reproduces theretention times actually measured. Based on the thus-adapted model“constants”, optimized parameters of the chromatography run(concentration pattern and/or temperature) can be calculated and setonce again. The optimized parameters calculated each time, the retentiontimes calculated therefrom and also the retention times then measuredwith the same set parameters are automatically recorded in adata-recording apparatus and stored arranged such that a substancemixture can clearly be allocated the corresponding iterative sequence ofchromatography runs, the set parameters and the measured values. The endof such a series is a chromatography run with optimized parameters, thecriterion chosen for definitively optimized parameters being e.g. thecondition that a maximum number of bands or peaks is recorded separatelyand a preset time limit is not exceeded, the time limit also being ableto be a time dependent on the number of recorded peaks. The data storagecan, if necessary, also be limited to the last set of optimizedparameters for a given substance mixture.

[0042] A further optimizing criterion chosen can be the condition thatthe changes in the retention times and/or parameters, which have beenobtained on the basis of two or more iterative chromatography runsdiffer only slightly from one another, so that it is to be expected thateven further iterative optimizations will deliver only slightimprovements.

[0043] The method according to the invention can run fully automaticallywith the exception of an initial inputting of known data, such as e.g.the known structural formulae of components of the substance mixture,the separation column type and any already-known retention times. Asalready mentioned, the essentially settable parameters are theconcentration of the organic modifier and the temperature.

[0044] If only a few components of the substance mixture are availableas standards, the retention times for the remaining components arecalculated and/or optimized in the model taken as a basis on thecondition that the parameter space covered by the components present asstandards is not exceeded. Such a parameter space contains e.g.restrictions in respect of the possible concentration of an organicmodifier because in every case the components (at least two) must berecorded within a preset time limit. Parameter conditions with which thetwo known components would not be clearly separable from one another arealso to be excluded. The parameters to be set then need still be soughtonly in the remaining range. The situation is analogous, of course, ifmore than two components are available as standards.

[0045] When carrying out the test runs and also during furtherchromatography runs in the course of the iterations, the number of peaksor bands of the chromatogram that are measured overall is also recorded.Starting from an ascertained maximum number of peaks, the parameters arefurther changed for optimization only inasmuch as the number of recordedpeaks does not fall as a result. This also necessitates considerablerestrictions of the available parameter space and thus a clearacceleration of the optimization.

[0046] To calculate and optimize the retention times, a so-calledpeak-tracking strategy is preferably used which is based on theidentification of peaks in a two-dimensional graph in which theretention times belonging to the peaks are represented as a function ofthe concentration of the organic modifier. It is understood that thisdoes not require a specific two-dimensional representation, but thatsuch a graph can also be purely mathematically abstractly recorded andanalyzed. In the process, the condition is used, among others, that twopeaks which have been recorded with different parameters are regarded asbelonging to the same component only if the retention time or thenatural logarithm of the retention time is a function that decreases asthe concentration of the organic modifier increases.

[0047] A further criterion for the identity of peaks is the similarityof the surfaces contained below a peak. Accordingly, two peaks areregarded as not identical or not belonging to the same component if therelative differences in the peak surfaces exceed a preset limit value ata given change in concentration of the organic modifier. Moreover, twopeaks can be regarded as identical if the spectral properties of thecomponents producing these peaks are identical or very similar.Accordingly, two peaks are regarded as not identical or not belonging tothe same component if the differences in the spectral properties exceeda preset limit value.

[0048] The method can be improved by also calculating the retentiontimes for at least one alternative stationary phase, i.e. an alternativecolumn material and optimizing them in relation to the variableparameters. This is expedient particularly if a satisfactory resultcannot be achieved for the initially considered column material, i.e.either disproportionately long retention times must be accepted or elsenot all the components of the substance mixture are recorded. In thiscase, another eluent can also be considered and a change made to adifferent chromatography column with the same or a different eluent. Themodel parameters ascertained based on the first column material and thefirst organic modifier or eluent can with certain restrictions betransferred to the model parameters for the new column material and thenew eluent or converted. The optimizing times can thereby likewise beconsiderably shortened for the second column material and/or the secondeluent. Expediently therefore, such transfers to differentchromatography columns or different eluents initially take placetheoretically only on the basis of the discussed model and a change thenpreferably takes place only if the optimizations carried out on thistheoretical basis promise a clear improvement vis-à-vis the initiallyused chromatography columns or eluents. With the method according to theinvention, the parameters, i.e. concentration of the organic modifier asa function of time or the temperature, are automatically set during thevarious chromatography runs which are optionally to be carried out inthe course of the optimization. A user need therefore only monitor thesystem or a corresponding chromatography system at certain intervals,the stored data expediently being recorded in the form of protocols andbeing shown on a screen or also another reproduction medium.

[0049] An artificial intelligence module is expediently used to decideon chromatography runs and the parameters to be set, which is e.g.capable of reaching compromises between the peak separation and theoverall duration. Such an artificial intelligence module can also beused for the optimization. In the search for optimum parameters forexample, a so-called Monte Carlo optimization can also be carried out.

[0050] The calculation of the retention times is carried out, dependingon whether certain data are known, according to different models. Onemodel, which is also called a solvatic model, is defined by the equationIn k′=a(V^(2/3))+b(ΔG)+c, k′ being the so-called capacity factor whichcorresponds to a retention time adjusted by the dead time, V being thevolume of the molecule of a sought component, ΔG the interaction energyof the molecule with water, and a, b and c parameters of thesorbent/eluent system. While a, b and c are obtained by calibrations ofsorbent/eluent systems, V and ΔG are adjusted in the course of theoptimizing process using the measured values. In addition, an empiricalmodel for the retention times can also be used simply by adaptationusing polynomials according to the equation In k′=a+bx+cx²+. . . , k′again being the previously mentioned capacity factor and x being definedas follows, depending on the chromatography method: Reversed-phasechromatography x = C Normal-phase chromatography x = In (C) Ion-exchangechromatography x = In (C) pH value of a buffer x = pH Temperature x =1/T

[0051] The coefficients a, b and c are model parameters which are to beset such that the known or measured values are correctly reproduced.Naturally, both models can also be applied combined with each other orindependently of one another to a given system, the solvatic modelnaturally being able to be used only if individual components or theirstructural formulae or molecule parameters are known. The mentionedmethod is further improved if an artificial intelligence module is usedwhich, on the basis of the results of the preceding experiments, allowsthe parameters for the next experiment to be set.

[0052] It is also assumed, without further statements, that a personskilled in the art can use the above description to the broadest extent.The preferred versions and examples are therefore to be understoodmerely as a descriptive, but in no way limiting, disclosure.

[0053] The full disclosure of all applications, patents and publicationslisted previously and hereafter, and also the corresponding applicationDE 101 28 546.9 are incorporated into this application by reference.

[0054] Further advantages, features and possible applications of thepresent invention follow from the following representation of anembodiment.

[0055] In principle, there are several different starting conditions.One initial situation is e.g. characterized in that the structuralformulae of at least some of the components are known. Retention timesfor a specific column type and an organic solvent can then be estimatedin accordance with the solvatic model. There follows a test run withcorrespondingly set conditions.

[0056] According to another scenario, no structural formulae whatsoeverare known for components and the substance mixture is essentiallyunknown. Here, a test can initially be carried out with a typical columnmaterial and a typical organic solvent. If this test is not successful,different column types and organic solvents are tried out, initiallyunder isocratic conditions, i.e. in each case with a fixed concentrationof an organic solvent, which concentration should not be chosen too low,and in addition two test runs should preferably be carried out, eachwith a clearly distinct concentration of the organic modifier.

[0057] FIG. 1 shows by way of example a chromatogram as was ascertainedfor seven sulphonamides under isocratic conditions at a firstconcentration of an organic modifier. However only 6 peaks are clearlydistinguishable. FIG. 2 shows the results for the same system, but witha second, lower concentration of an organic modifier. Here also only 6peaks are clearly recognizable. The double structure of the first peakin FIG. 2 and also the irregularities in the second peak in FIG. 1indicate however that at least one further analyte, which has not yetbeen dissolved in this chromatogram, is hidden in the substance mixture.

[0058] If structural formulae are known, the solvatic retention model istaken as a specific basis. If the structures are known, the systemcalculates the values for V and for ΔG automatically according to knownmethods (1. S. V. Galushko, J. of Chromatography, 552 (1991), 91-102; 2.S. V. Galushko, A. A. Kamenchuk, G. L. Pit, J. of Chromatography A, 660(1994) 47-59). The parameters a, b and c which are determined by thesolvent or the eluent and the stationary phase are obtained by acalibration with a known standard system from substance mixtures orindividual components. For further calculation, the parameters a, b andc then remain unchanged, so that the retention times result directlyfrom the structural formulae. Due to the concentration dependency of theparameters a, b and c, the optimum concentrations for a substancemixture comprising several components can now also be calculated if allthe components are to be recorded separately from one another within areasonable time.

[0059] A test run is then carried out with the correspondinglycalculated parameters. The test results are then used for the refinementof the model parameters and a new calculation of retention times untilthe model agrees with the times measured in reality. The then-optimizedmolecule parameters V and ΔG and also the set parameters for an optimumchromatography run are then stored for future use.

[0060] If the structures are not known, the empirical model is used. Afirst run is initially carried out under isocratic conditions. Then asecond run is carried out with a different concentration of organicmodifier, the second concentration being chosen either clearly higher orclearly lower depending on the results of the first run. After thesecond run has also been carried out and the data are recorded, if alinear model is used initially, i.e. in the polynomial mentioned aboveIn k′=a+bx+cx²+ . . . only the coefficients a and b are assumed to beother than 0.

[0061] Optimized conditions are calculated anew from this and a furthertest run carried out. On the basis of the three measured values thenavailable in total, the quadratic model can be used, i.e. the parameterc is now also assumed to be other than 0. A further optimization takesplace starting from that previously ascertained; optimum isocraticconcentrations by ascertaining an optimum gradient profile. The systemcan produce a large number of corresponding gradient profiles accordingto the random principle and in each case calculate, with the previouslyascertained, optimized model parameters, the associated chromatogram orthe position of the bands of the individual components, wherein asuitable optimization criterion (e.g. the product of the intervalsbetween all the adjacent peaks divided by the square of the longestretention time) is to be selected and the optimum profile can then beascertained, using so-called “Monte Carlo methods”, according to therandom principle. Additional boundary conditions such as e.g. thecondition that the concentration can only be constant or increasing,facilitate the discovery of an optimum gradient profile.

[0062] As a result at least part of the following data is obtained inthe course of an optimization:

[0063] 1 ) Optimum isocratic conditions,

[0064] 2) Optimum conditions with linear gradient,

[0065] 3) Different gradient profiles with gradient varying step-wisebased on the different retention models.

[0066] It transpires that the optimum conditions are very often at timesisocratic conditions, the transfer from an initially low concentrationto a higher concentration taking place with a very steep gradient andalmost sharply.

[0067] The time required for an optimization naturally depends on thenumber of components or compounds to be analyzed. 10-12 hours areusually required to optimize the conditions for a substance mixture withfive components on a conventional column. If the molecule parameters Vand ΔG are known, the optimization can take place even more quickly. Themethod according to the invention and the corresponding device haveabove all the advantage that they proceed automatically without externalmeasures by operators as soon as a few parameters have been inputted andthe system has been started.

[0068] An embodiment is represented below which shows the automatedoptimization of a chromatography of seven sulphonamides using only twostandards. The two standards were sulfathiazole and sulfameracil.Purospher STAR RP18e was used as sorbent, 0.005 m phosphate bufferpH=3.5 as eluent.

[0069] Detection at 270 nm

[0070] Temperature 30° C.

[0071] Flow rate 1.0 ml/min

[0072] Injection volume 10 μl

[0073] Dead time at 0.4 minutes

[0074] Washing time on new solvent 6.0 minutes

[0075] Starting concentration 80%

[0076] Delay time 1.25 minutes.

[0077] Nine successive test runs were carried out for sulfathiazole andsulfameracil under these test conditions, the concentration of theeluent being reduced from run to run, starting from 80% through 60, 40,25, 19, 13, 8 to 6%, a further run being carried out at 12%. Each time,the time until the passage of a peak was measured and in each case thepeak resolution factors and the factor K′_(max) measured, i.e. themaximum standardized duration (T−TO)/TO with T=retention time andTO=dead time.

[0078] Both peaks were found down to concentration of 8% in each case,the resolution factor RS improving continuously from 0.08 through 0.19,0.82, 0.96, 1.62, 2.46 to 3.82. A further reduction of the concentrationto 6% resulted in the peak for sulfathiazole not being found. Anincrease in the concentration compared with the previous 8% led to aslight reduction of the resolution factor and to a lowering of themaximum standardized retention time k′_(max).

[0079] Accordingly the best isocratic conditions had been measured at aconcentration of 8%. Thereafter runs were carried out with the wholemixture. An elimination criterion was a peak surface of 30,000 in unitareas (e.g. μVs). In 18 runs, the concentration was reduced step-wisefrom 80% through 71%, 63%, 55%, 49%, 45%, 35%, 28%, 25%, 22%, 19%, 16%,13%, 10%, 7%, 5% and raised again to 8% and 12%. The retention times,the peak widths, the peak surfaces and the number of theoretical platesof the peaks were ascertained. Likewise the pair resolution and also thestandardized retention time were also ascertained from thechromatograms. It transpired that an isocratic concentration of 19%delivered the best values.

[0080] Only then are e.g. linear retention models built up, taking intoaccount e.g. the surface, the retention time and spectral data of thepeaks.

[0081] Elution with an 80-% concentration for approximately 250 secondsis always carried out between each of the runs.

[0082] Two of the chromatograms which resulted from the aforementionedtests are shown in the already discussed FIGS. 1 and 2, FIG. 2 showingthe optimum for isocratic conditions. The system them modifies the testruns by setting gradients of the eluent. For this for example a linearretention model is built up which takes into account the measuredretention times, the peak surfaces and the spectral data. On this basis,multi-stage gradients are sought and used in further runs. Examples ofthis are shown in FIGS. 3, 4 and 5.

[0083] It is essential that the chromatography conditions are chosensuch that all seven peaks which were found in previous tests areobtained in every case, appear within an acceptable time and are easilyseparable from one another and able to be resolved. An essentialcriterion is therefore, in addition to the number of peaks, theparameter of pair resolution. For the gradient pattern shown in FIG. 3,in addition to the clearly recognizable seven peaks, a parameter of pairresolution of 3.511 is obtained for the critical peak pair and a maximumretention time of approximately 17.5 minutes. In the case of thegradient pattern according to FIG. 4, a maximum retention time of just19 minutes, but also an improved parameter of pair resolution of 4.341,results. A further improved pair resolution with a value of 4.506 isobtained for the gradient pattern as finally represented in FIG. 5. Theascertained maximum retention time of 19.2 minutes is in every casestill in an acceptable range with the result that the chromatographyconditions represented in FIG. 5 (gradient pattern) in the presentexample were selected as optimum after further test runs yielded nobetter pair resolution and at most a slight reduction of the maximumretention time.

1. Method for the automatic performance of chromatographicinvestigations with the help of an electronic data processing apparatusand with the following steps i) Recording of data which include at leastone of the following items of information a) Retention times of at leastone component of the substance mixture, preferably for at least twodifferent concentrations of an organic modifier, b) The temperature atwhich the retention times were ascertained, c) Structural formulae ofone or more of the components of the substance mixture, d) Type ofeluent and also the concentration of an organic modifier therein, e)Parameters or type of separation column, the recorded data being fullyor partly able to be retrieved and/or supplemented from a data storageapparatus and, where data are unavailable for the items of informationaccording to a), b) or c), at least a first test run taking place underisocratic conditions in order to record retention times according to a),and preferably a further isocratic test run being carried out with adifferent concentration of the organic modifier, ii) Calculation ofretention times as a function of optimizing parameters based on a modelwhich reproduces a functional relationship of at least part of the datarecorded under i), the recorded data also being able to be reproduced bysetting model parameters, iii) Optimization of the retention times inthe model by varying the optimizing parameters, iv) Carrying out atleast one further chromatography run with the parameters of theretention times optimized according to step iii), and comparing theactual retention times with the theoretically calculated, optimizedretention times, v) Optionally single or repeated repetition of steps i)to iv) if the deviations of the calculated and the measured retentiontimes exceed a presettable limit value or if the differences between theretention times of two successive chromatography runs fall short of apresettable limit value, the decision for parameters to be specificallyset for a next run preferably being taken by an artificial intelligencemodule.
 2. Method according to claim 1, wherein on the basis of themanually inputted initial data such as e.g. the structural formulae ofcomponents of the substance mixture, the separation column type andalready known retention times and parameters, steps i) to v) proceedautomatically, with the exception of an initial inputting of data. 3.Method according to claim 1, characterized in that the ascertainedoptimized values, preferably including the values ascertained inintermediate steps, are stored in a database.
 4. Method according toclaim 1, characterized in that before step ii) the recorded data arecompared with stored data.
 5. (Currently Amended) Method according toclaim 1, characterized in that the concentration of the organicmodifier, the pH value and the temperature T are used as optimizingparameters.
 6. Method according to claim 1, characterized by the stepFixing of a maximally available parameter space by test runs with atleast two standards using the condition that the standards can bedetected separately from one another within a presettable maximum time.7. Method according to claim 1, a maximum number of peaks being usedadditionally as an optimization criterion in step iii).
 8. Methodaccording to claim 1, characterized in that, to calculate and optimizethe retention times, a peak-tracking strategy is used which is based onthe identification of peaks in a two-dimensional graph in which theretention times belonging to the peaks are represented compared with theoptimizing parameters.
 9. Method according to claim 8, that peaks areallocated to one another as identical only if, for their connecting linein the two-dimensional representation retention time vs concentration ofthe organic modifier, the following is true: d InT/dC<0, C being theconcentration of the organic modifier.
 10. Method according to claim 8,characterized in that peaks are regarded as not identical if therelative deviations of the peak surfaces of a seemingly identicalcomponent exceed a preset limit value.
 11. Method according to claim 8,characterized in that peaks are not regarded as identical if deviationsof spectral parameters exceed a preset limit value.
 12. Method accordingto claim 1, which furthermore includes the step that the retention timesare calculated and/or optimized in relation to the variable parametersfor at least one alternative stationary phase (alternative columnmaterial).
 13. Method according to claim 12, characterized in that anautomatic change to a different column and/or a different eluent takesplace if the model predicts that the optimization criteria will bebetter fulfilled thereby than with the first column material and/oreluent.
 14. Method according to claim 1, characterized in that presetdata and parameters and also the associated retention times, the numberof peaks and the peak surfaces for all test runs and optimizedchromatography runs are stored assigned to one another if they relate tothe same system (substance mixture, eluent, column).
 15. Methodaccording to claim 1, characterized in that the inputted data and/orresults of a run are compared with the stored data, and, if the dataagree within preset limits, the stored data of the best run for thissystem are used for the next run.
 16. Device for the automaticperformance and optimization of chromatographic investigations with a) Achromatography separation column, b) An inlet chamber for the substancemixture, c) A feed apparatus for the eluent, d) A detection andmeasuring unit at the outlet of the chromatography column, e) Adata-recording unit for the automatic and/or manual recording of data,characterized in that f) the data-recording unit is allocated anoptimization unit which calculates, on the basis of the most recentlyrecorded data of a system, optimized parameters for a (further)chromatography run and g) a control apparatus and parameter sensors areprovided, the control apparatus being connected to the optimization unitsuch that the calculated optimized parameters are automatically set bythe control apparatus and an automatic chromatography run takes placewith this setting.
 17. Device according to claim 16, characterized inthat, for the optimization and/or decision for optimized data, includesa module that operates according to the principles of artificialintelligence.
 18. Device according to claim 16, characterized in that inthe search for optimized parameters a so-called Monte Carlo optimizationis carried out.
 19. Device according to claim 18, characterized in thata module is provided which evaluates the criteria maximum number ofpeaks, separation of peaks and overall time limit of a parameterselection, and deduces specific parameters therefrom.
 20. Deviceaccording to claim 1, characterized in that change apparatuses areprovided for the transfer to a different column material and/or adifferent eluent.